The factors controlling delta Gs of electron and proton transfers in the bacterial photosynthetic reaction center (RC) will be studied. The RC is the best characterized of the intramembrane election transfer proteins that store chemiosmotic energy in the cell by coupling electron and proton transfers. A combination of experimental and theoretical approaches will be used to understand how interactions between cofactors and protein specify the related properties of cofactor affinity for a binding site, in AM electrochemistry, and pKa's of surrounding residues. Classical electrostatics calculations will be used to analyze electrochemical midpoints (E-m's) of the different redox cofactors in RCs. E-m's of active and inactive sides of the RC will be calculated to determine if electron transfer could be limited by delta G differences. Optical measurement of flash initiated electron transfer will place limits on the rate of charge recombination from the secondary quinone along the inactive side of the protein. Modification of the quinone and binding site will determine if this reaction is limited by local effects or by the intervening protein. For the two quinone binding sites in RCs, the picture of factors controlling E-m's obtained by electrostatics calculations will be compared with detailed calculations of quinone and semiquinone affinities for the site. This analysis will complement a rich data base of measured binding constants and Em's. The function of quinones with different charge distributions will be characterized experimentally and theoretically. The Boltzmann distribution of different protonation states in RCs will be obtained by Monte Carlo sampling of the electrostatic energies of different ionization states to determine residue pKas. pKa shifts in different redox states will show the pathway of proton uptake that is coupled to electron transfer. The perturbation of calculated pKa's and E-m's because of polar proton motions coupled to electron transfer will be explored. This will be complemented by experimental measurement of the temperature dependence of the delta G between (BChl)2+BPh- and (BChl)2+QA- to investigate if the processes that yield the substantial relaxation of (BChl)2+BPh- on the ns to mu s time scale can be frozen out and characterized.